The enzymatic hydrolysis of cellulose to glucose has gained increased interest over the last ten years, and growing demand for economically sustainable biofuels points to an urgent need for reducing costs in their production. Cellulose, a polysaccharide made by many plants, is one of the most abundant organic compounds on Earth and therefore represents a potential goldmine for the biofuel industry. However, current enzymatic degradation of cellulose faces major issues that prevent its wide utilization to produce economically competitive biofuel.
Cellulose is hydrolyzed to glucose via the synergistic action of several enzymes. Endoglucanases (E.C. 3.2.1.4) break down cellulose chains at random positions within the cellulose chains whereas exoglucanases (i.e. cellobiohydrolases, E.C. 3.2.1.91) cleave off cellobiose specifically from the chain ends in a processive manner. Cellobiose is subsequently converted into glucose by β-glucosidase (E.C. 3.2.1.21). The exo-endo synergism is easily understood by the fact that endoglucanases provide more chain ends for cellobiohydrolases to act upon. The hydrolysis of insoluble, solid cellulose is a heterogeneous reaction, which cannot be described by kinetic models based just on Michaelis-Menten kinetics. After an initial phase of adsorption of cellulases on cellulose (fast comparatively to hydrolysis), the enzymes cleave off cellobiose and move along the same chain, hydrolyzing glycosidic bonds until an event that terminates cleavage occurs. As the reaction proceeds to intermediate degrees of conversion, the rate decreases dramatically, and the last part of the cellulose hydrolysis requires an inordinate amount of overall total reaction time. Several factors, both substrate- and enzyme-related, have been suggested to be responsible for this slowdown of rate, but so far no clear picture of what is limiting the reaction has been proposed. The substrate characteristics often implied in the slowdown of rate include surface area, porosity, degree of polymerization, crystallinity, and overall composition (complex substrates such as lignocellulosics vs. pure cellulose). For enzyme-related features, deactivation, inhibition, jamming, clogging, and imperfect processivity are often advanced as causes for slowdown.
One of the most controversial theories concerns the influence of crystallinity and the change of the degree of crystallinity during enzymatic hydrolysis. It is accepted that the initial degree of crystallinity of cellulose plays a major role as rate determinant in the hydrolysis reaction. A completely amorphous sample is hydrolyzed much faster than a partially crystalline cellulose, which has led to the currently widespread assumption that amorphous domains in a partially crystalline cellulose sample are hydrolyzed first, leaving crystalline parts to be hydrolyzed at the end, thus resulting in an increased crystallinity index and explaining the dramatic drop in rate at higher degrees of conversion.
However, studies of this phenomenon differ by the analytical methods (X-ray diffraction vs. solid state 13C-NMR), the nature of the substrate used (complex lignocellulosics vs. pure cellulose) and the source of the hydrolytic enzymes (mostly from Trichoderma reesei and other fungal strains). Several reviews have stated that it is still difficult to conclude that crystallinity is a key determinant of the rate of enzymatic hydrolysis. See, for example, (1) Lynd, L. R.; Weimer, P. J.; van Zyl, W. H.; Pretorius, I. S., Microbial cellulose utilization: Fundamentals and biotechnology. Microbiology and Molecular Biology Reviews 2002, 66, (3), 506−+; (2) Zhang, Y. H. P.; Lynd, L. R., Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems. Biotechnology and Bioengineering 2004, 88, (7), 797-824; and (3) Mansfield, S. D.; Mooney, C.; Saddler, J. N., Substrate and enzyme characteristics that limit cellulose hydrolysis. Biotechnology Progress 1999, 15, (5), 804-816. Usually, different types of cellulose with different degrees of crystallinity were employed in these studies (such as cotton, cotton linter, Avicel, filter paper, or bacterial cellulose). Their cellulase-catalyzed degradation gave hydrolysis rates directly related to the initial crystallinity index of the cellulose sample. To correctly relate crystallinity index with hydrolysis rate, it is of prime importance to study samples that have the same basic composition and provenance. More importantly, pure cellulose is superior to complex substrates, as the presence of lignin or hemicellulose may interfere with the cellulase action and reduce accessibility (and therefore hydrolysis rates).
Another important criteria related to hydrolysis rate involves adsorption capacity of cellulases onto cellulose. The rate of hydrolysis has been shown to be proportional to the amount of adsorbed enzymes. Additionally the amount of adsorbed endoglucanases was found to be strictly related to the hydrolyzability of crystalline cellulose. Furthermore, the degree of crystallinity of cellulose influences adsorption at a given protein loading and maximum adsorption constant has been shown to be greatly enhanced with low crystallinity indexes. The same work concluded that the effective binding was the limiting parameter in hydrolysis rate in the case of cellulose with low degrees of crystallinity (despite high adsorption constant).
Amorphous cellulose is a favored modification to investigate cellulase activity. Obtained after treatment with 85% phosphoric acid, the cellulose obtained in this way, PASC (phosphoric acid swollen cellulose), results from complete dissolution of the sample and the treatment was shown to have no impact on the reducing-end concentration of the cellulose sample (i.e. its degree of polymerization). The effect of various phosphoric acid concentrations has only been investigated across a narrow range of acid concentrations or mainly at low concentrations. Recently, Zhang et al. showed that the concentration of phosphoric acid used to generate swollen cellulose dictates the rate of enzymatic hydrolysis by controlling the state of cellulose solubilization (Biomacromolecules 2006, 7, (2), 644-648).
Accordingly, there remains a need for determining the main causes of rate limitations in the enzymatic hydrolysis of, for example, Avicel and other biomass materials, and especially the role of, for example, crystallinity and adsorption on cellulose susceptibility to enzymatic degradation. Both 13C-NMR solid-state spectroscopy and X-ray crystallography may be applied to investigate the crystallinity of pure cellulose (Avicel) at different degrees of conversion by cellulases from Trichoderma reesei, the most commonly studied cellulase-producing organism. Cellulose (Avicel) with controlled degrees of crystallinity may be generated with phosphoric acid solutions of precisely calibrated concentration. These cellulose samples from the same source may be employed to investigate and elucidate the relationship between degree of crystallinity, adsorption and enzymatic hydrolysis rates.
In general terms the instant invention pertains in one embodiment to a method for assessing value of a biomass in an enzymatic hydrolysis comprising first measuring an initial hydrolysis rate of the biomass and then correlating the measured initial hydrolysis rate with an established crystallinity index of standard cellulosic material to project a crystallinity indicative of overall enzymatic hydrolysis susceptibility.
In another embodiment the instant invention generally pertains to a method for producing glucose for fermentation. The method comprises:
(a) treating a biomass with acid and heat under conditions sufficient to produce a composition mixture comprising cellulose suitable for enzymatic hydrolysis;
(b) enzymatically hydrolyzing at least a portion of the cellulose of step (a) under conditions sufficient to form a composition comprising glucose; and
(c) fermenting glucose by, for example, utilizing glucose in an aerobic or anaerobic fermentation process. Typically, one or more reaction conditions of steps (a), (b), or (c) are selected by first measuring an initial hydrolysis rate of said biomass and then selecting one or more appropriate reaction conditions based upon said initial hydrolysis rate.
In yet another embodiment, the invention pertains to a method of using the initial hydrolysis rate to improve the efficiency of an enzymatic hydrolysis. The method comprises:
(a) measuring the initial hydrolysis rate of a biomass to be subjected to hydrolysis and correlating the measured initial hydrolysis rate with an established crystallinity index of standard cellulosic material to project a crystallinity indicative of overall enzymatic hydrolysis susceptibility;(b) selecting a hydrolysis enzyme and determining the required amount of said enzyme to produce cellulosic sugars with said biomass;(c) determining the overall hydrolysis rate at said required amount of said enzyme;(d) selecting or adjusting one or more of the following enzymatic reaction conditions: pre-treatment step, reaction time, reaction temperature, type of enzyme, and amount of enzyme; and(e) enzymatically hydrolyzing a biomass using one or more of said selected conditions.